Diabetic kidney disease (DKD) is the most common cause of end-stage kidney disease in the US, and podocyte injury is an important feature of DKD. However, mechanisms of podocytopenia in DKD are poorly understood. Podocyte apoptosis and activation of RhoA are observed in immortalized human podocytes exposed to the sera of patients with T1D or T2D and DKD in conjunction with cholesterol accumulation and can be prevented by cyclodextrin (CD), an oligosaccharide that removes cholesterol from cells and that protects diabetic mice from the development of proteinuria. Excessive glomerular cholesterol deposition has been described in clinical and experimental DKD, is associated with impaired cholesterol efflux as suggested by the down-regulation of ATP-binding cassette transporter (ABCA1) expression, and can be induced by TNFa. The relative contribution of free and esterifed cholesterol to podocyte injury is unknown. Our long-term goal is to develop therapeutic strategies that target podocyte cholesterol accumulation in DKD. We hypothesize that: 1) TNFa-mediated downregulation of ABCA1 in podocytes results in accumulation of cholesterol; 2) accumulation of esterified cholesterol in early DKD causes podocyte hypertrophy while free cholesterol in late DKD causes podocyte apoptosis; 3) therapeutic strategies that reduce intracellular cholesterol accumulation protect podocytes in DKD. In order to test these hypotheses, we will utilize genetically modified mouse models, NIH approved models of DKD and in vitro experiment in human podocytes with the aim to: 1-Determine if ABCA1 deficiency in podocytes worsens podocyte injury and proteinuria in DKD. 2-Determine the relative contribution of free and esterified cholesterol to podocyte injury. 3-Determine if podocytopenia in DKD can be prevented by podocyte depletion of free cholesterol with CD and/or ABCA1 inducers. This proposal is innovative as the role of intracellular free and esterified cholesterol n the pathogenesis of podocyte injury in DKD has never been explored. In order to match innovation with adequate expertise, we have assembled a unique team of investigators that includes podocyte biologists, lipid biologists, pathologists, nephrologists and endocrinologists. We aim to advance the knowledge related to DKD by uncovering novel disease mechanisms amenable to therapeutic intervention.